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Time-resolved FRET between GPCR ligands reveals oligomers in native tissues

Nature Chemical Biology volume 6, pages 587–594 (2010)Cite this article

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Abstract

G protein–coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.

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Figure 1: FRET signal between antagonists bound to V1a or oxytocin receptors expressed in heterologous expression systems.
Figure 2: FRET signal between antagonists bound to dopamine D2 receptors expressed in heterologous expression systems.
Figure 3: Binding experiments on the oxytocin receptor expressed in membrane preparations from native tissue.
Figure 4: FRET signals between fluorescent antagonists or agonists bound to native oxytocin receptors expressed in rat mammary glands.
Figure 5: FRET experiments performed on patches of native tissues.

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Acknowledgements

Thanks are due to S. Granier, P. Rondard and L. Prezeau for their critical reading of the manuscript. This work was supported by research grants from the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Actions Concertées Incitatives “Molécules Cibles et Thérapeutiques” (no. 240 and 355), ANR-06-BLAN-0087-03 and ANR-09-BLAN-0272) and the US National Institutes of Health (grants GM025280, DA022413, MH54137). This work was also made possible by the Plateforme de Pharmacologie-Criblage of Montpellier and the Region Languedoc-Roussillon. NAPS and PPHT amines were synthesized by the National Institute of Mental Health Chemical Synthesis and Drug Supply Program and provided by A.N. and J.J.

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Author notes

  1. Laura Albizu & Michaela Kralikova

    Present address: Present addresses: Department of Neurology, Mount Sinai School of Medicine, New York, New York, USA (L.A.); Department of Auditory Neuroscience, Czech Academy of Science, Prague, Czech Republic (M.K.).,

  2. Laura Albizu and Martin Cottet: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Montpellier, France

    Laura Albizu, Martin Cottet, René Seyer, Isabelle Brabet, Jean-Philippe Pin, Bernard Mouillac & Thierry Durroux

  2. Institut National de la Santé et de la Recherche Médicale, Montpellier, France

    Laura Albizu, Martin Cottet, René Seyer, Isabelle Brabet, Jean-Philippe Pin, Bernard Mouillac & Thierry Durroux

  3. Université Montpellier 1 and 2, Montpellier, France

    Laura Albizu, Martin Cottet, René Seyer, Isabelle Brabet, Jean-Philippe Pin, Bernard Mouillac & Thierry Durroux

  4. Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Michaela Kralikova, Marie-Laure Rives & Jonathan Javitch

  5. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Michaela Kralikova, Marie-Laure Rives & Jonathan Javitch

  6. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA

    Michaela Kralikova, Marie-Laure Rives & Jonathan Javitch

  7. Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, Ohio, USA

    Stoytcho Stoev & Maurice Manning

  8. Cisbio Bioassays, Bagnols sur Cèze, France

    Thomas Roux, Hervé Bazin, Emmanuel Bourrier, Laurent Lamarque & Eric Trinquet

  9. Département de Physiologie, Laboratoire de Neuroendocrinologie du Développement, Université des Sciences et Technologies de Lille, Villeneuve d′Ascq, France

    Christophe Breton

  10. Medicinal Chemistry Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland, USA

    Amy Newman

  11. Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Jonathan Javitch

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Contributions

L.A. and T.D. originated the project. L.A., M.C. and T.D. executed most of the experiments and wrote the manuscript; S.S. and M.M. performed the peptide agonist synthesis; R.S. synthesized the antagonist peptide; I.B. performed experiments with SNAP-tag receptors; M.-L.R., M.K., A.N. and J.J. contributed to the development of the fluorescent dopamine receptor ligands and assay system; T.R. characterized the dopamine ligands; H.B., E.B. and L.L. labeled the ligands with fluorophores; C.B. performed saturation experiments with [125I]OTA; E.T., B.M. and J.-P.P. supported the project and participated in the writing of the manuscript.

Corresponding authors

Correspondence to Jean-Philippe Pin, Bernard Mouillac or Thierry Durroux.

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Competing interests

T.R., H.B., E.B., L.L.and E.T. are employees of Cisbio, which develops HTRF compatible fluorescent ligands and products combining its HTRF technology with Covalys SNAP-tag protein labeling system, and therefore may gain financially through publication of this paper. Part of the work of CNRS UMR 5203 has been financially supported by Cisbio and by an unrestricted grant from Senomyx.

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Albizu, L., Cottet, M., Kralikova, M. et al. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol 6, 587–594 (2010). https://doi.org/10.1038/nchembio.396

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